This invention relates to novel semisynthetic antifungal compounds which are prepared by the acylation of the cyclic peptide nucleus produced by the enzymatic deacylation of antibiotic A30912 factor A.
Antibiotic A-30912 factor A is an antifungal cyclic peptide having the formula: ##STR9## wherein R is the linoleoyl group ##STR10## Throughout this application, the cyclic peptide formulas, such as formula I, assume that the amino acids represented are in the L-configuration. The factor is isolated from the A30912 complex which contains other factors arbitrarily designated factors B, C, D, E, and G. The A-30912 complex and the individual factors A through G are described by M. Hoehn and K. Michel in U.S. Pat. No. 4,024,245. Factor A is identical to antibiotic A-22802 which is described by C. Higgins and K. Michel in U.S. Pat. No. 4,024,246. Factor A has also been found to be identical to antibiotic echinocandin B [see F. Benz et al., Helv. Chim. Acta, 57, 2459 (1974) and Swiss Pat. No. 568,386] and to antibiotic SL 7810/F [see C. Keller-Juslen et al. Tetrahedron Letters, 4147 (1976) and Belgium Pat. No. 834,289].
Antibiotic A-30912 factor A is prepared by fermentation using one of several different organisms, namely: (a) Aspergillus rugulosus NRRL 8113 (see U.S. Pat. No. 4,024,245); (b) Aspergillus nidulans NRRL 8112 (see U.S. Pat. No. 4,024,246); (c) Aspergillus nidulans var. echinulatus A-32204 as described in Swiss Pat. No. 568,386; (d) Aspergillus rugulosus NRRL 8039 (see Belgian Pat. No. 834,289); or (e) Aspergillus nidulans var. roseus NRRL 11440 (see co-pending application of L. Boeck and R. Kastner, METHOD OF PRODUCING THE A-30912 ANTIBIOTICS, Ser. No. 126,078, filed Mar. 3, 1980, which is a continuation-in-part of application Ser. No. 46,744, filed June 8, 1979, (now abandoned), the entire disclosure of which is incorporated herein by reference.)
A subculture of A. nidulans var. roseus has been deposited and made a part of the permanent culture collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Peoria, Ill. 61604, from which it is available to the public under the number NRRL 11440.
When a strain of A. nidulans var. roseus NRRL 11440 is used to produce A-30912 factor A, a complex of factors is obtained which for convenience is called the A-42355 antibiotic complex. A-30912 factor A is the major factor of the A-42355 antibiotic complex, while factors B, D and H are minor factors. Examples 96, 97, and 98, herein, illustrate the preparation of the A-42355 complex and the isolation and purification of A-30912 factor A therefrom. A-30912 factor H is further described in a co-pending application of Karl. H. Michel entitled ANTIBIOTIC A-30912 FACTOR H, Ser. No. 117,739, filed Feb. 1, 1980, which is a continuation-in-part of application Ser. No. 46,875, filed June 8, 1979 (now abandoned).
In the A-30912 factor A molecule (Formula I), the linoleoyl side chain (R) is attached at the cyclic peptide nucleus at the .alpha.-amino group of the dihydroxyornithine residue. Surprisingly, it has been found that the linoleoyl side chain can be cleaved from the nucleus by an enzyme without affecting the chemical intregity of the nucleus. The enzyme employed to effect the deacylation reaction is produced by a microorganism of the family Actinoplanaceae, preferably the microorganism Actinoplanes utahensis NRRL 12052, or a variant thereof. To accomplish deacylation, antibiotic A30912 factor A is added to a culture of the microorganism and the culture is allowed to incubate with the substrate until the deacylation is substantially complete. The cyclic nucleus thereby obtained is separated from the fermentation broth by methods known in the art. Unlike antibiotic A-30912 factor A, the cyclic nucleus (lacking the linoleoyl side chain) is substantially devoid of antifungal activity.
The cyclic nucleus afforded by the aforedescribed enzymatic deacylation of antibiotic A-30912 factor A, is depicted in Formula II. ##STR11##
Removal of the side chain group affords a free primary .alpha.-amino group in the dihydroxyornithine residue of the cyclic peptide. For convenience, the compound having the structure given in Formula II will be referred to herein as "A-30912A nucleus." As will be apparent to those skilled in the art, A-30912A nucleus can be obtained either in the form of the free amine or of the acid addition salt. Although any suitable acid addition salt may be employed, those which are non-toxic and pharmaceutically acceptable are preferred.
The method of preparing A-30912A nucleus from antibiotic A-30912 factor A by means of fermentation using Actinoplanes utahensis NRRL 12052 is described in the co-pending application of Bernard J. Abbott and David S. Fukuda, entitled "A-30912A NUCLEUS", Ser. No. 103,017, which was filed Dec. 13, 1979. A continuation-in-part application of this application, with the corresponding Ser. No. 103,149, is being filed herewith this even date, the full disclosure of which is incorporated herein by reference. Example 93, herein, illustrates the preparation of A-30912A nucleus by fermentation using antibiotic A-30912 factor A as the substrate and Actinoplanes utahensis NRRL 12052 as the microorganism.
The enzyme produced by Actinoplanes utahensis NRRL 12052 may be the same enzyme which has been used to deacylate penicillins (see Walter J. Kleinschmidt, Walter E. Wright, Frederick W. Kavanagh, and William M. Stark, U.S. Pat. No. 3,150,059, issued Sept. 22, 1964).
Cultures of representative species of Actinoplanaceae are available to the public from the Northern Regional Research Laboratory under the following accession numbers:
Actinoplanes utahensis: NRRL 12052 PA1 Actinoplanes missouriensis: NRRL 12053 PA1 Actinoplanes sp.: NRRL 8122 PA1 Actinoplanes sp.: NRRL 12065 PA1 Streptosporangium roseum var. hollandensis: NRRL 12064 PA1 --CH.sub.2 --; ##STR20## in which R.sup.5 is C.sub.1 -C.sub.4 alkyl (i.e., methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, or 1-methylpropyl); --(CH.sub.2).sub.m in which m is an integer from 2 to 10; and ##STR21## in which p is an integer from 1 to 8 and q is an integer from 0 to 7, provided that n+m must be no greater than 8. PA1 (a) CH.sub.3 --; PA1 (b) --(CH.sub.2).sub.n CH.sub.3 wherein n is an integer from 1 to 16; and ##STR22## wherein r and s are independently, an integer from 0 to 14 provided that r+s can be no greater than 14. Illustrative C.sub.2 -C.sub.17 alkenyl radicals, which are preferred for the purpose of this invention, are PA1 (a) --(CH.sub.2).sub.t --CH.dbd.CH--(CH.sub.2).sub.u --CH.sub.3 wherein t and u are independently, an integer from 0 to 14 provided that t+u can be no greater than 14. PA1 (b) --(CH.sub.2).sub.v --CH.dbd.CH--(CH.sub.2).sub.y --CH.dbd.CH--(CH.sub.2).sub.z --CH.sub.3 wherein v and z are independently, an integer from 0 to 11 and y is an integer from 1 to 12 provided that v+y+z can be no greater than 11. PA1 CH.sub.3 -- PA1 CH.sub.3 (CH.sub.2).sub.5 -- PA1 CH.sub.3 (CH.sub.2).sub.6 -- PA1 CH.sub.3 (CH.sub.2).sub.8 -- PA1 CH.sub.3 (CH.sub.2).sub.10 -- PA1 CH.sub.3 (CH.sub.2).sub.12 -- PA1 CH.sub.3 (CH.sub.2).sub.14 -- PA1 CH.sub.3 (CH.sub.2).sub.16 -- PA1 cis-CH.sub.3 (CH.sub.2).sub.5 CH.dbd.CH(CH.sub.2).sub.7 -- PA1 trans-CH.sub.3 (CH.sub.2).sub.5 CH.dbd.CH(CH.sub.2).sub.7 -- PA1 cis-CH.sub.3 (CH.sub.2).sub.10 CH.dbd.CH(CH.sub.2).sub.4 -- PA1 trans-CH.sub.3 (CH.sub.2).sub.10 CH.dbd.CH(CH.sub.2).sub.4 -- PA1 cis-CH.sub.3 (CH.sub.2).sub.7 CH.dbd.CH(CH.sub.2).sub.7 -- PA1 trans-CH.sub.3 (CH.sub.2).sub.7 CH.dbd.CH(CH.sub.2).sub.7 -- PA1 cis-CH.sub.3 (CH.sub.2).sub.5 CH.dbd.CH(CH.sub.2).sub.9 -- PA1 trans-CH.sub.3 (CH.sub.2).sub.5 CH.dbd.CH(CH.sub.2).sub.9 -- PA1 cis, cis-CH.sub.3 (CH.sub.2).sub.4 CH.dbd.CHCH.sub.2 CH.dbd.CH(CH.sub.2).sub.7 -- PA1 trans, trans-CH.sub.3 (CH.sub.2).sub.4 CH.dbd.CHCH.sub.2 CH.dbd.CH(CH.sub.2).sub.7 -- PA1 cis,cis,cis-CH.sub.3 CH.sub.2 CH.dbd.CHCH.sub.2 CH.dbd.CHCH.sub.2 CH.dbd.CH--(CH.sub.2).sub.7 --.
The effectiveness of any given strain of microorganism within the family Actinoplanaceae for carrying out the deacylation of this invention is determined by the following procedure. A suitable growth medium is inoculated with the microorganism. The culture is incubated at about 28.degree. C. for two or three days on a rotary shaker. One of the substrate antibiotics is then added to the culture. The pH of the fermentation medium is maintained at about pH 6.5. The culture is monitored for activity using a Candida albicans assay. Loss of antibiotic activity is an indication that the microorganism produces the requisite enzyme for deacylation. This must be verified, however, using one of the following methods: (1) analysis by HPLC for presence of the intact nucleus; or (2) reacylation with an appropriate side chain (e.g. linoleoyl, stearoyl, or palmitoyl) to restore activity.
It is known that other antibiotic substances possess the same nucleus as that of antibiotic A-30912 factor A. These antibiotics differ from antibiotic A-30912 factor A in that different acyl groups are present in place of the linoleoyl group (R) in Formula I. Such antibiotics are: (a) tetrahydro-A-30912 factor A (tetrahydro-SL 7810/F; tetrahydro echinocandin B) described in Belgium Pat. No. 834,289 and by F. Benz et al., Helv. Chim. Acta, 57 2459 (1974), which compound is depicted in Formula I when R is stearoyl; and (b) aculaecin A, which is a component of the aculaecin complex (prepared by fermentation using Aspergillus aculeatus NRRL 8075) and is described by K. Mizuno et al., in U.S. Pat. No. 3,978,210. As is discussed in Belgium Pat. No. 859,067, in aculaecin A the palmitoyl side chain is present in place of linoleoyl. Tetrahydro-A-30912 factor A can be prepared from antibiotic A-30912 factor A by catalytic hydrogenation using PtO.sub.2 in ethanol under positive pressure. Both tetrahydro-A-30912 factor A and aculaecin A can be employed as substrates for the enzymatic deacylation using the procedures herein described.